The anaerobic protists, Giardia lamblia and Entamoeba histolytica, are enteric Category B pathogens that each infect more than a billion people worldwide. They are credible bioterrorism threats as fewer than ten cysts are needed for human infection and cysts can readily be added to water supplies. Both cause debilitating diarrhea in normal adults. There is a great need for improved treatments as -15% of giardial isolates are resistant to metronidazole (Mz), a 5-nitroimidazole (Nl), that is the current drug of choice. Because of its relatively high efficacy, safety, and low cost, we have elected to use Mz as a lead compound for drug discovery. We have already synthesized new 5-NI derivatives with increased activity against Mz-resistant Giardia. We will now test the hypothesis that novel derivatives at three critical positions of the 5-NI core structure will lead to credible drug candidates with increased activity against diverse Mz-sensitive and Mz-resistant Giardia. We will apply these findings to Entamoeba later in the project. Our Specific Aims are: AIM 1. Identification of next-generation 5-Nls with increased potency against Mz-sensitive and Mz-resistant Giardia in vitro. We will evaluate the potency of compounds prepared by traditional synthetic organic chemistry and by the innovative and powerful "click" chemistry strategy, using rapid initial screens to identify compounds with the highest activity against Giardia and Entamoeba. Activity screens and syntheses will follow an iterative process of lead optimization. AIM 2 is to evaluate the efficacy of carefully selected next generation 5-Nls in animal models of giardiasis. We will evaluate the most potent new compounds identified in Aim 1 in murine models, and will then test the four most efficacious drugs in feline giardiasis. AIM 3 is to define giardial mechanisms of activation of and resistance to new 5-NI drugs. We will also develop new Giardia lines resistant to the novel 5-NI compounds and employ them in further drug development in Aim 1. Our ultimate goal is to produce at least two credible novel candidate 5-NI drugs, suitable for commercial development, that are effective against important anaerobic pathogens and will have great benefit to public health worldwide and to defense against bioterrorism.